The common approach to identification of disease causing mutations usually involves screening at a level of genomic DNA or cDNA. There are several methods which can detect single base substitutions in a single or double stranded DNA segment. These include RNAse sensitivity assay [Myers et al., Science 230:1242-1246 (1985)], denaturing gradient gel electrophoresis [Myers et al., Nature 313:495-498 (1985)], chemical cleavage at mismatches [Cotton et al., Proc Natl Acad Sci USA 85:4397-4401 (1988)] and, finally, single strand conformation polymorphism analysis [Orita et al., Genomics 5:874-879 (1989)]. Once the aberrant fragment is identified, the mutation is verified by sequencing. These methods detect both the pathogenic mutations as well as the normally occurring polymorphisms. Eventually, the detected changes need to be tested for pathogenicity by expression in E. coli [Gregersen et al, Hum Genet 86:545-551 (1991)] or mammalian cells [e.g., Reichardt et al., Genomics 12:596-600 (1992)]. These methods do not distinguish between nonpathogenic and pathogenic mutations or allow simultaneous localization and expression of the mutation. Therefore, such screening methods require weeks of effort before the pathogenic mutation can be identified. What is needed is a rapid screening method that allows simultaneous localization and expression of the pathogenic mutation.
Cystathionine .beta.-synthase (CBS) [L-serine hydrolyase (adding homocysteine); EC 4.2.1.22], a pyridoxal 5'-phosphate (PLP) dependent enzyme, plays a key role in the transsulfuration of homocysteine in eukaryotes [Mudd et al., The Metabolic Basis of Inherited Disease, 6th ed., pp 693-734, McGraw-Hill, New York (1989)]. Under physiological circumstances, about half of the intracellular homocysteine is transsulfurated yielding cysteine. The remainder is remethylated to methionine in the methionine cycle [Finkelstein and Martin, J Biol Chem 259:9508-9513 (1984)]. S-Adenosyl-L-methionine (AdoMet) regulates homocysteine flux through these branches. At low levels of AdoMet, remethylation of homocysteine is unimpaired. Elevated levels, on the other hand, inhibit remethylation pathways and increase the irreversible transsulfuration by stimulating CBS [Kutzbach and Stockstad, Biochim Biophys Acta 250:459-477 (1971); Finkelstein and Martin, Biochem Biophys Res Commun 118:14-19 (1984); Finkelstein et al., Biochem Biophys Res Commun 66:81-87 (1975)].
Deficiency of CBS is the leading cause of homocystinuria (HCS) in humans. The symptoms and signs of this condition are well characterized. They include dislocated optic lenses, skeletal disorders, mental retardation, and often fatal thromboembolism [Mudd et al., The Metabolic Basis of Inherited Disease, 6th ed., pp 693-734, McGraw-Hill, New York (1989)]. Transsulfuration is blocked, resulting in the accumulation of homocyst(e) ine and often methionine together with depletion of cysteine and cystathionine in body fluids. In about half of the affected patients, these symptoms can be alleviated by pyridoxine administration. The rationale for this treatment is based, in part, on the observation that some of the mutant CBS proteins bind pyridoxal 5'-phosphate less avidly than normal [Lipson et al., J Clin Invest 66:188-193 (1980) ].
Normal CBS is a homotetramer of 63 kDa subunits [Skovby et al., J Biol Chem 259:583-593 (1984)]; its gene resides on human chromosome 21 at q22.3 [Skovby et al., Hum Genet 65:291-294 (1984); Munke et al., Am J Hum Genet 42:550-559 (1988)]. It has been shown that CBS mutations do not usually alter the subunit size; however, the intracellular CBS concentrations are generally markedly reduced [Skovby et al., Am J Hum Genet 36:452-459 (1984)]. These studies suggest that the majority of the synthase genetic defects are missense rather than nonsense mutations or internal deletions. None of these mutations has been examined at the molecular level. To facilitate these studies of CBS mutations, a rapid screening method for pathogenic mutations was developed incorporating either a prokaryotic or eukaryotic expression system. Such an expression screening system has wide applicability in detecting and localizing pathogenic mutations involved in diseases which include genetically inherited diseases. Some examples of genetically inherited diseases include glutaric acidemia type I caused by a deficiency of glutaryl-CoA dehydrogenase, glutaric acidemia type II caused by a deficiency of ETF dehydrogenase, acyl-CoA dehydrogenase medium chain deficiency, propionic acidemia caused by a deficiency of propionyl-CoA carboxylase, Sandhoff's disease characterized by a defect in the production of hexosaminidases A and B, citrullinemia which is a disease of the amino acid metabolism caused by a deficency in argininosuccinic acid synthetase, and Fabry's disease, an X-linked recessive disorder due to deficiency of .alpha.-galactosidase.